The ultimate goal of this research renewal is to develop a series of nutritionally complete prepared diets that will reduce the costs of production and will increase the numerical and temporal availability of cephalopods, which are carnivorous and now require expensive live diets. Cephalopods are prime models for studies of the nervous system, cellular biophysics, memory, vision, circulatory system, equilibrium reception, metabolism and behavior. The future use of cephalopods as sophisticated invertebrate substitutes for modem vertebrate-oriented biomedical research should increase in response to directives made by the U.S. Congress and National Research Council. During the first three years of this grant, major milestones toward the development of prepared diets have been attained: (1) behavioral studies determined that cephalopods (octopuses, cuttlefishes and squids) can learn or be induced through routine laboratory methods to ingest pelleted diets, (2) chemotaxis by octopuses has been demonstrated for the first time with several chemicals (proline, ATP and crab extract) and (3) adult octopuses and both juvenile and adult, cuttlefishes have been maintained or grown on pelleted diets for up to 60 days. As a result, we are closer to a pelleted diet than originally proposed and the last major impediment to the economical culture of cephalopods has been partially overcome. Improvements in the nutritional quality and nutrient availability of the diets remain as mandatory unresolved issues for both cuttlefishes and squids, requiring a comprehensive nutritional approach. The proposed novel and integrated strategy (i.e., stepwise analysis of orientation, ingestion, digestion, utilization and growth) is now proven and guarantees the successful formulation of prepared diets for cephalopods of all ages. The identification of chemical attractants and feeding incidents for cuttlefishes and squids will improve feeding responses and ingestion rates. Palatability studies will focus on the relationship between chemical and textural stimuli and their effect on feeding. The form and textural stability of the diets will be especially important for cuttlefishes and squid since they eat while swimming. Analysis of individual feed ingredients and formulated diets will provide information on the nutrient availability of such major components as protein, energy and lipid. A series of growth trials will evaluate the influence of protein/energy ratio and protein quality (amino acid balance) on growth survival, food conversion ratio and protein efficiency ratio. The effects of protein quality and lipid levels of the diets on digestive enzyme synthesis and secretion will be assayed with specific substrates for protease, carbohydrase and lipolytic enzyme activities. Finally, the pelleted diets will be presented to squids in a production-scale system using time-controlled automatic or demand feeders. The proposed research is significant because it: (1) provides basic and applied information on the dietary requirements of cephalopods, (2) develops standard laboratory diets that can be used by any laboratory in the world and (3) dramatically decreases costs while concurrently increasing the availability of cephalopods for biomedical research.